Biochemical and genetic approaches will be used to study actin assembly in yeast. Principles established are likely to apply to more complex eukaryotes where actin dynamics underlie diverse cellular processes and where defective cytoskeleton function contributes to conditions such as muscular dystrophy, certain hereditary anemias, and cancer. The following aims will be pursued: (1) Actin nucleotide-binding pocket mutant. To test physiological importance of actin ATP hydrolysis and Pi release, and to genetically identify regulators of filament stability, we will take advantage of a unique actin mutant. The V159N mutation uncouples the actin nucleotide cycle from filament destabilization. Specifically, we will test the role of rapid actin filament turnover in pheromone-induced cellular morphogenesis and cortical actin patch motility, and will use the V159N mutant to genetically identify regulators of actin dynamics. (2) Biochemical and structure-function analysis of the cofilin-actin interaction. Our isolation of yeast cofilin, elucidation of its three dimensional molecular structure, and demonstration that it promotes actin filament disassembly in vivo, provide a strong foundation for further studies of this important and ubiquitous protein. The molecular models of yeast cofilin and actin filaments will now be docked, providing novel insights into the mechanism of cofilin-promoted filament disassembly. To more fully elucidate steps regulating the assembly/disassembly cycle, we will identify factors which stimulate formation of ATP-actin monomers from ADP-actin:cofilin complexes formed during disassembly. (3) Genetic analysis of cofilin regulation and regulation of actin filament stability. In response to regulatory signals, changes in actin assembly typically occur on time scales that mandate regulation by second messengers and posttranslational modification. Since genetic approaches provide powerful avenues to elucidation of regulatory pathways, proteins which regulate cofilin will be identified by genetic suppression. These studies are important for determining how cells trigger rapid cytoskeletal rearrangements required for diverse processes including morphogenesis and cytokinesis.